Rapid formation of poly-γ-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro

D. W. Fry, J. C. Yalowich, I. D. Goldman

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Abstract

The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate, 4-amino-10-methylpteroylglutamate (4-NH2-10-CH3-PteGlu1), and their interactions with dihydrofolate reductase were evaluated in the Ehrlich ascites tumor cell in vitro by high pressure liquid chromatography. The accumulation of these metabolites was increased over 5-fold by the addition of 5 mM L-glutamate or L-glutamine and exhibited a positive correlation with the extracellular concentration of methotrexate. Derivatives having from 1 to 5 glutamyl residues (4-NH2-10-CH3-PteGlu(2-6)) were detected and accumulation occurred in a distinctive pattern. 4-NH2-10-CH3-PteGlu2 appeared rapidly and reached a steady state level within 60 min which persisted over 4 h. The appearance of 4-NH2-10-CH3-Pte-Glu3 and -Glu4 was detected after short lag periods and their levels continued to increase for at least 4 h becoming the predominant derivatives within the cell. Higher derivatives (4-NH2-10-CH3-PteGlu5 and -Glu6) were detected after 1 h; however, their levels remained low. As the levels of intracellular methotrexate polyglutamate derivatives increased, there was a decline in intracellular methotrexate. Studies employing gel filtration shown that this was due, at lest in part, to the replacement of methotrexate on dihydrofolate reductase by its metabolites and the subsequent efflux of the previously bound methotrexate from the cell. The percentage of polyglutamate derivatives bound to dihydrofolate reductase was similar to the percentage of non-bound derivatives, indicating a high affinity of these metabolites for this enzyme. When polyglutamate derivatives were in excess of the dihydrofolate reductase binding capacity and extracellular methotrexate was removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the majority of the enzyme. Although methotrexate polyglutamates were detected in the extracellular compartment, a chemical gradient (inside/outside) of over 200 was maintained across the membrane. These studies indicate that intracellular methotrexate is rapidly converted to poly-γ-glutamyl derivatives, these metabolites effectively compete with metotrexate for binding on dihydrofolate reductase, and these derivatives are retained within the cell more effectively than methotrexate.

Original languageEnglish (US)
Pages (from-to)1890-1896
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number4
Publication statusPublished - Jan 1 1982
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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